LOCUS       sequence-396574-        5514 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescence control plasmid for selecting ncAA-encoding 
            Methanomethylophilus alvus Pyl-RS mutants. Expresses sfGFP-WT and M.
            alvus Pyl-tRNA(6). p15a origin of replication.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5514)
  AUTHORS   Avila-Crump S, Hemshorn ML, Jones CM, Mbengi L, Meyer K, Griffis JA,
            Jana S, Petrina GE, Pagar VV, Karplus PA, Petersson EJ, Perona JJ, 
            Mehl RA, Cooley RB
  TITLE     Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA 
            Synthetases for Structurally Diverse Non-Canonical Amino Acids.
  JOURNAL   ACS Chem Biol. 2022 Dec 16;17(12):3458-3469. doi: 
            10.1021/acschembio.2c00639. Epub 2022 Nov 16.
  PUBMED    36383641
REFERENCE   2  (bases 1 to 5514)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acschembio.2c00639"; journalName: "ACS Chem Biol"; date: 
            "2022-12-16- 16"; volume: "17"; issue: "12"; pages: "3458-3469"
COMMENT     Sequence Label: pALS2-sfGFP WT
FEATURES             Location/Qualifiers
     source          1..5514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(534..550)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(543..565)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             895..939
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(977..995)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      991..1038
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1209..2000)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2001..2092)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2107..2124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2241..2786)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2321..2340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3074..3952)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3979..4263
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     4186..4205
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             join(4297..4299,4300..4302,4303..5013)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             5020..5037
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5116..5133)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5286..5372
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5464..5491
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
       61 aatggtgcag ggcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
      121 gttgggcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
      181 ggcgacgata gtcatgcccc gcgcccaccg gaaggagcta ccggcagcgg tgcggactgt
      241 tgtaactcag aataagaaat gaggccgctc atggcgttct gttgcccgtc tcactggtga
      301 aaagaaaaac aaccctggcg ccgcttcttt gagcgaacga tcaaaaataa gtggcgcccc
      361 atcaaaaaaa tattctcaac ataaaaaact ttgtgtaata cttgtaacgc tagatctggg
      421 ggacggtccg gcgaccagcg ggtctctaaa acctagcata gcggggttcg acaccccggt
      481 ctctcgccaa ctagtatcct tagcgaaagc taaggatttt ttttaagctt ggcactggcc
      541 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
      601 gcacatcccc ctttcgccag acgctctccc ttatgcgact cctgcattag gaagcagccc
      661 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagccc
      721 gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctggcggccg
      781 cataatgctt aagtcgaaca gaaagtaatc gtattgtaca cggccgcata atcgaaatta
      841 aattggatat cggcaggcca cgcgatcgct gacgtcggta ccctcgagtc tggtaaagaa
      901 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
      961 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1021 gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt
     1081 ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc
     1141 cctgaaccga cgaccgggtc atcgtggccg gatcttgcgg cccctcggct tgaacgaatt
     1201 gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct
     1261 tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta
     1321 gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg
     1381 acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc
     1441 actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca
     1501 tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga
     1561 cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg
     1621 atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc
     1681 agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact
     1741 tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg
     1801 atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata
     1861 tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac
     1921 gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg
     1981 gcgatcaccg cttccctcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2041 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aatagctagc
     2101 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     2161 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     2221 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     2281 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2341 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     2401 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     2461 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     2521 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     2581 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     2641 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     2701 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     2761 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     2821 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     2881 agttgtaatt ctcatgttag tcatgccccg cgcccaggat ctcaccgtca tcaccgaaac
     2941 gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc
     3001 aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct
     3061 gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg
     3121 cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt
     3181 gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa
     3241 gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact
     3301 gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg
     3361 cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc
     3421 gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt
     3481 gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa
     3541 tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc
     3601 ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa
     3661 agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct
     3721 ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt
     3781 tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc
     3841 ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat
     3901 gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca
     3961 tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact
     4021 gcgtctttta ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc
     4081 tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg
     4141 gcagaaaagt ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt
     4201 tatccataag attagcggat cctacctgac gctttttatc gcaactctct actgtttctc
     4261 catacccgtt ttttgggcta acaggaggaa ttaaccatgg ttagcaaagg tgaagaactg
     4321 tttaccggcg ttgtgccgat tctggtggaa ctggatggtg atgtgaatgg ccataaattt
     4381 agcgttcgtg gcgaaggcga aggtgatgcg accaacggta aactgaccct gaaatttatt
     4441 tgcaccaccg gtaaactgcc ggttccgtgg ccgaccctgg tgaccaccct gacctatggc
     4501 gttcagtgct ttagccgcta tccggatcat atgaaacgcc atgatttctt taaaagcgcg
     4561 atgccggaag gctatgtgca ggaacgtacc attagcttca aagatgatgg cacctataaa
     4621 acccgtgcgg aagttaaatt tgaaggcgat accctggtga accgcattga actgaaaggt
     4681 attgatttta aagaagatgg caacattctg ggtcataaac tggaatataa tttcaacagc
     4741 cataatgtgt atattaccgc cgataaacag aaaaatggca tcaaagcgaa ctttaaaatc
     4801 cgtcacaacg tggaagatgg tagcgtgcag ctggcggatc attatcagca gaataccccg
     4861 attggtgatg gcccggtgct gctgccggat aatcattatc tgagcaccca gagcgttctg
     4921 agcaaagatc cgaatgaaaa acgtgatcat atggtgctgc tggaatttgt taccgccgcg
     4981 ggcattaccc acggtatgga tgaactgtat aaaggcagcc accatcatca tcaccattaa
     5041 agctcgagat ctgcagctgg taccatatgg gaattcgaag cttggctgtt ttggcggatg
     5101 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
     5161 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     5221 gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
     5281 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     5341 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     5401 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     5461 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctg tcga
//
